Our proposal is to develop a platform of R-type pyocins as bactericidal agents that can be engineered to be remarkably pathogen-specific, while versatile enough to enable rapid modification of their specificity towards a diverse range of pathogenic bacteria. Specifically, we will generate a battery of highly specific therapeutic and prophylactic agents targeting STEC serogroups O26, O45, O55, O91, O103, O111, O113, O118, O121, O145, O146, as well as Salmonella enterica serogroup D1, including serotypes enteriditis and typhi, and Vibrio cholera serotypes O1 and O139. The work proposed is based on our findings that R-type pyocins are highly specific and highly potent bactericidal protein complexes that have been shown to be efficacious for systemic bacterial infections in animals. While highly specific to their target organism, we have shown that these bactericidal agents can be retargeted to other bacteria by modifying their tail fibers with the related tail proteins of bacteriophages. We therefore hypothesize that R-type pyocins can be used as a versatile platform to target a broad range of human pathogenic bacteria. The ubiquity and diversity of phages specific against virtually every type of bacteria, promise broad utility for this platform. In order to achieve our goals, we will first isolate Podoviridae bacteriophages that recognize and kill the pathogen of interest and identify its tail fiber (Specific Aim #1). That tail fiber will then be fused to a truncated native tail fiber of the R2-type pyocin and expressed together with the remaining pyocin genome, creating a viable pyocin with altered specificity against the pathogen of interest (Specific Aim #2) This portfolio of potent, targeted bactericidal agents will have broad applications in the management of the public health and clinical aspects of widespread infection by these enteric pathogens. These specific therapeutic agents could be used to manage accidental or intentional, large- or small scale infections. Due to their extraordinary specificity, they are expected to eliminate the target pathogens while leaving normal, healthy, microflora intact. Importantly, given the diversity of bacteriophages available against a wide array of bacteria, this versatile platform can be developed for specificities far beyond what is proposed herein, have far wider applicability against nearly all known bacteria. PUBLIC HEALTH RELEVANCE: We propose to develop a versatile, pure protein-based platform for defense against a diverse array of dangerous bacteria that cause severe intestinal infections and potentially death. Our platform, based on altering the binding and killing specificity of the basic R-type pyocin structure by attaching only the specific tail proteins of bacterial viruses, offers a potent response to a wide range of pathogens which pose a threat to public health in the form of accidental or intentional food- or waterborne outbreak.